Method and apparatus for simultaneously analyzing vanillylmandelic acid, homovanillic acid and creatinine

ABSTRACT

A method for analyzing vanillylmandelic acid, homovanillic acid and creatinine by fast liquid chromatography, which comprises a step of introducing a test solution containing vanillylmandelic acid, homovanillic acid and creatinine to a cation exchange resin column and separating creatinine from vanillylmandelic acid and homovanillic acid; a step of separating by a separation column vanillylmandelic acid and homovanillic acid in the test solution from which creatinine has been separated, and a step of detecting each component.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Neuroblast tumor (hereinafter referred to simply as NB) is a malignanttumor specific to infancy and curable only when discovered at an earlystage. Therefore, early discovery and diagnosis are desired. In the caseof true disease, vanillylmandelic acid (hereinafter referred to simplyas VMA) and homovanillic acid (hereinafter referred to simply as HVA) asmetabolic products of catechol amine are discharged into urine insubstantial amounts. Accordingly, it is possible to diagnose NB in ahigh probability by measuring these two components in urine. In Japan,NB mass screening for children has been started since January 1985 at agovernmental level.

The present invention relates to a method for simultaneously analyzingVMA, HVA and creatinine (hereinafter referred to simply as CRN) in atest solution quickly and accurately with high sensitivity for such NBmass screening, and to an apparatus useful for such a method.

2. DISCUSSION OF BACKGROUND

In recent years, it has been common to use fast liquid chromatographyfor the measurement of VMA and HVA. In this method, the separation isconducted by reversed phase chromatography or anion exchangechromatography and the detection is conducted by a ultravioletabsorption measuring method (A. Yoshida, et al, J. Chromatogr., 227,162(1982)), an electrochemical measuring method (M. H. Joseph, et al, J.Chromatogr., 226,361 (1981)) or a fluorescence measuring method (T. G.Rosano, et al, Clin. Chem. 27,228 (1981)).

However, in the test solution, there exist acidic components such asuric acid and other organic acids and basic components such aspolyamines, in addition to the object components. These inclusions arelikely to interfere with the determination. Accordingly, it used to bedifficult to directly inject the test sample, and it used to be requiredto have some pretreatment conducted. For such pretreatment, it has beencommon to employ a method wherein urine or urine-absorbed filter paperis extracted with ethyl acetate or with a buffer solution. However, eachof such methods involves manual operation and thus has drawbacks suchthat not only the operation is cumbersome but also the recovery ratesare likely to be irregular, and it is not possible to completelyeliminate the influence of inclusions.

On the other hand, in the determination of the quantity of a certaincomponent in urine, it is common to adopt a creatinine ratio (CRN ratio)as a means for correction against the change of the amount of urine.Likewise, in the quantitative determination of VMA and HVA, it isnecessary to preliminarily measure the CRN concentration in urine. Inthe conventional methods, the measurement of CRN was conducted by amethod entirely independent from the system for the measurement of VMAand HVA, whereby the analysis of data was not only complex but alsotime-consuming. Therefore, the simplification has been desired.

As mentioned above, the conventional methods require a cumbersomepretreatment operation such as solvent extraction of VMA and HVA.Further, since the CRN analysis used to be impossible in a single seriesof measuring system, it took a long period of time for the overallanalysis.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method foranalyzing VMA, HVA and CRN faster and more accurately with highersensitivity than such conventional methods, and to provide an apparatusfor analysis to be used for such a method.

The present invention provides a method for analyzing vanillylmandelicacid, homovanillic acid and creatinine by fast liquid chromatography,which comprises a step of introducing a test solution containingvanillylmandelic acid, homovanillic acid and creatinine to a cationexchange resin column and separating creatinine from vanillylmandelicacid and homovanillic acid; a step of separating by a separation columnvanillylmandelic acid and homovanillic acid in the test solution fromwhich creatinine has been separated; and a step of detecting eachcomponent.

Further, the present invention provides an apparatus for analyzingvanillylmandelic acid, homovanillic acid and creatinine by fast liquidchromatography, which comprises a cation exchange resin column (7A) forseparating creatinine from vanillylmandelic acid and homovanillic acidin a test solution, an anion exchange column (7B) for partiallyfractionating vanillylmandelic acid and homovanillic acid from otheracidic components, a separation column (9) for separatingvanillylmandelic acid and homovanillic acid, a flow path switching valve(8A) connected to the cation exchange column (7A) to change the path ofeluent, a flow path switching valve (8B) connected to the anion exchangeresin column (7B) and the separation column (9), and a detector (10, 11)for detecting each component, wherein the cation exchange resin column(7A) and the anion exchange resin column (7B), and the anion exchangeresin column (7B) and the separation column (9) are connected in series,respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view illustrating an embodiment of theapparatus useful for analysis according to the method of the presentinvention.

FIG. 2 illustrates the sequential operation of the apparatus foranalysis in the Example.

FIG. 3 is a chromatogram as an example of the analysis of a standardsample and a test sample.

DETAILED DESCRIPTION OF THE INVENTION

Now, the present invention will be described in detail.

The cation exchange resin column for separating CRN from VMA and HVAaccording to the present invention is preferably a column packed with astrong acid type cation exchange resin. VMA, HVA and other acidiccomponents have no interaction with such a column, whereas CRN and otherbasic components have an interaction therewith. Accordingly, VMA and HVAare eluted first, whereby they are separated from CRN.

Then, the test solution from which CRN has been separated, is led to theseparation column for separating VMA and HVA. As such a separationcolumn, a reversed phase chromatography column or an anion exchangeresin column may be employed. It is preferred to employ the reversedphase chromatography column.

In order to maintain the useful life of the searation column, it iseffective that prior to the separation of VMA and HVA, partialfractionation is conducted by an anion exchange resin column, preferablyby a weak base type anion exchange resin column, to partiallyfractionate VMA and HVA from other acidic components so that unnecessaryacidic components other than VMA and HVA will not be introduced to theseparation column. Here, the partial fractionation means that VMA andHVA may not necessarily completely be separated from other acidiccomponents.

Various methods may be employed for the detection of the respectivecomponents for the present invention. For the detection of CRN, it ispreferred to employ an ultraviolet absorption measuring method. Whereas,for the detection of VMA and HVA, an ultraviolet absorption measuringmethod, an electrochemical measuring method or a fluorescence measuringmethod may suitably be selected. However, from the viewpoint of thesensitivity and selectivity, the electrochemical measuring method or thefluorescence measuring method is preferred.

As the eluent to be used in each separation step of the presentinvention, there may be employed a phosphoric acid buffer solutioncontaining acetonitrile, which is commonly employed.

By adopting the above-mentioned method, not only VMA and HVA but alsoCRN can be separated and analyzed simultaneously by a single fast liquidchromatography apparatus by using a single test sample solution.

The analytical method of the present invention and the analyticalapparatus to carry out the method will be further described withreference to the drawings.

FIG. 1 shows an embodiment of the method and apparatus of the presentinvention.

This analytical apparatus comprises a cation exchange resin column (7A)for separating CRN from VMA and HVA in the test solution, an anionexchange resin column (7B) for partially fractionating VMA and HVA fromother acidic components, a separation column (9) for separating VMA andHVA, a valve (8A) connected to the cation exchange resin column (7A), avalve (8B) connected to the anion exchange resin column (7B) and theseparation column (9), a detector (10) for detecting VMA and HVA and adetector (11) for detecting CRN, wherein said cation exchange resincolumn (7A) and said anion exchange resin column (7B), and said anionexchange resin column (7B) and said separation column (9) are connectedin series, respectively. Further, the apparatus includes eluent tanks(1A-1E), eluent switching valves (2A, 2B), eluent supplying pumps (4A,4B), a degasser (3), a sample supplying device (5), prefilters (6A, 6B)and a waste solution tank (12).

The test solution which is a sample, is led by the eluent (1A) to thecation exchange resin column (7A), where VMA, HVA and other acidcomponents eluting first will be separated from CRN and other basiccomponents eluting later. The eluent from which CRN has been separated,is then led to the anion exchange resin column, where VMA and HVA arepartially fractionated from other acidic components. VMA and HVA elutingfirst through the anion exchange resin column (7B) are then led to aseparation column (9) by an eluent (1C, 1D) by switching the valve (8B).Here, VMA and HVA are separated. CRN eluting later from the cationexchange resin column (7A) is led to a detector (11) by the valveoperation and thereby detected. On the other hand, VMA and HVA elutingfrom the separation column (9) are likewise detected by a detector (10).After the completion of the detection of the respective components, therespective eluents (1B, 1E) are supplied to wash the eluent paths. Aftercompletion of this initial operation, the analysis may be repeated byintroducing a sample again.

The combination or sequential order of the cation exchange resin column(7A), the anion exchange resin column (7B) and the separation column (9)may be different from the above, so long as the cation exchange resincolumn (7A) and the anion exchange resin column (7B), and the anionexchange resin column (7B) and the separation column (9) are connectedin series, respectively.

It is advantageous to control such an analytical apparatus by a computerfrom the viewpoint of the analytical operation and saving of the timefor the analysis.

In FIG. 1, a six way valve is used as the valve (8A). However, the valve(8A) may be a four way valve.

For the detection of CRN, it is possible to use a third pump for thecation exchange resin column (7A). For the simplification, however, theanalytical apparatus shown in FIG. 1 has only two supplying pumps.

Now, the present invention will be described in further detail withreference to an Example. However, it should be understood that thepresent invention is by no means restricted by such a specific Example.

EXAMPLE

The analytical apparatus used for the analysis was as shown in FIG. 1,wherein as the cation exchange resin column (7A), a strong acid-type TSKGEL SP-2 SW (4.6 I.D. x 120 mm, manufactured by TOSOH CORPORATION) wasused, as the anion exchange resin column (7B), a weak base-type TSK GELDEAE-2 SW (4.6 I.D. x 120 mm, manufactured by TOSOH CORPORATION) wasused, and as the separation column (9), TSK GEL ODS-80 TM (4.6 I.D. x150 mm, manufactured by TOSOH CORPORATION) for reversed phasechromatography was used. For the detection of VMA and HVA, afluorescence detector was used as the detector (10), and the wavelengthfor measurement was 280 nm for λEX and 320 nm for λEM. For the detectionof CRN, an ultraviolet absorption detector was used as the detector(11), and the wavelength (λ) for the measurement was 240 nm.

With respect to various eluents, a 50 mM phosphoric acid buffer solution(pH 4.8) containing 10% of acetonitrile was used as the eluent (1A), a50 mM phosphoric acid buffer solution (pH 4.8) containing 10% ofacetonitrile and 300 mM sodium chloride was used as the eluent (1B), a50 mM phosphoric acid buffer solution (pH 5.2) was used as the eluent(1C), a 50 mM phosphoric acid buffer solution (pH 5.2) containing 6% ofacetonitrile was used as the eluent (1D) and a 50 mM phosphoric acidbuffer solution (pH 5.2) containing 50% of acetonitrile was used as theeluent (1E).

As the test sample solutions, a standard sample containing 4 ng of eachof VMA and HVA and 400 ng of CRN, and urine discharged by a healthy manby single urination, were used.

The operational sequence for carrying out the analytical operation willbe described with reference to FIG. 2.

Firstly, the paths for eluents are set for the initial state. As shownin FIG. 2(a), the eluent (1C) is filled in the detector (10) through theseparation column (9). On the other hand, the eluent (1A) is filled inthe other path. The sample is introduced from the sample supplyingdevice (5) to the eluent path, and VMA, HVA and CRN are led to thecation exchange resin column (7A). Here, when VMA and HVA eluting firstare introduced to the anion exchange resin column (7B), the valve (8A)is switched as shown in FIG. 2(b) to temporarily stop the supply to thecation exchange resin column (7A) and stop the elution of CRN. When thepartial fractionation in the anion exchange resin column (7B) with theeluent (1A) has been completed and VMA and HVA have eluted, the valves(8A, 8B) are switched as shown in FIG. 2(c), whereupon VMA and HVA areled by the eluent (1C) to the separation column (9), while CRN is led tothe detector (10) by the eluent (1A) and thereby detected. In theseparation column (9), VMA and HVA are separated by the eluent (1C) andthen by the eluent (1D) as shown in FIG. 2(d). Then, the valve (8B) isswitched as shown in FIG. 2(e) not to introduce other acidic componentspartially fractionated in the anion exchange resin column (7B) to theseparation column (9). At the same time, the eluent (1B) is supplied tothe path to which the eluent (1A) has been supplied, to wash the path,and then the valve is switched as shown in FIG. 2(f) to supply theeluent (1A) for the initial state. Meantime, VMA and HVA are completelyseparated and detected by the detector. After the separation anddetection, the path of the separation column (9) is washed by the eluent(1E) as shown in FIG. 2(g), and then finally switched to the eluent (1C)as shown in FIG. 2(h) for the initial state.

The analyses of the standard sample and the urine test sample wereconducted by controlling the above analytical operation by a computer.The eluents flowing through the respective columns during each step andthe operational step of each valve are shown in Table 1 together withthe operational sequence of FIG. 2. One cycle for the introduction of asample was 15 minutes.

                                      TABLE 1                                     __________________________________________________________________________    Time (min.)                                                                         Column (7A)                                                                          Column (7B)                                                                          Column (9)                                                                          Column (8A)                                                                          Column (8B)                                                                          FIG. 2                                __________________________________________________________________________    0.0-2.2                                                                             1A     1A     1C    OFF    OFF    a                                     2.2-6.5                                                                             (1A)   1A     1C    ON     OFF    b                                     6.5-9.0                                                                             1A     1C     1C    OFF    ON     c                                     9.0-10.0                                                                            1A     1D     1D    OFF    ON     d                                     10.0-10.5                                                                           1B     1B     1D    OFF    OFF    e                                     10.5-14.0                                                                           1A     1A     1D    OFF    OFF    f                                     14.0-14.5                                                                           1A     1A     1E    OFF    OFF    g                                     14.5-15.0                                                                           1A     1A     1C    OFF    OFF    h                                     __________________________________________________________________________

The chromatogram thus obtained is shown in FIG. 3.

As is apparent from the foregoing description, the present inventionprovides the following advantageous:

(1) In the analyses of VMA and HVA and CRN, a cumbersome operation suchas a pretreatment operation for the extraction with ethyl acetate, isnot required;

(2) VMA, HVA and CRN can be simultaneously analyzed, whereby the CRNratio important as an index for the NB diagnosis can readily andaccurately be measured; and

(3) No special or complex operation is required for the separation ofeach component, and the analyses can be completed in a short period oftime. The operation can be readily controlled by a computer, wherebymanpower can be saved.

What is claimed is:
 1. A method for analyzing a test solution comprisingvanillylmandelic acid, homovanillic acid and creatinine by fast liquidchromatography, which comprises:a step of introducing a first inletstream comprising a test solution containing vanillylmandelic acid,homovanillic acid and creatinine and a second inlet stream comprising afirst eluent to a cation exchange resin column, separating creatininefrom vanillylmandelic acid and homovanillic acid using said firsteluent, and removing from said cation exchange resin column a firstoutlet stream comprising vanillylmandelic acid and homovanillic acid anda second outlet stream comprising creatinine, wherein at least one ofsaid first and second outlet streams further comprises said firsteluent; a step of introducing said first outlet stream from said cationexchange resin column and a second eluent stream into a separationcolumn, separating vanillylmandelic acid and homovanillic acid usingsaid second eluent, and removing from said separation column a thirdoutlet stream comprising vanillylmandelic acid and a fourth outletstream comprising homovanillic acid, wherein at least one of said thirdand fourth outlet streams further comprises said second eluent; and astep of detecting each component in said second, third and fourth outletstreams.
 2. A method of detecting neuroblastoma by analyzing a testsolution according to the method of claim 1 and comparing the amount ofvanillylmandelic acid and homovanillic acid to those of a normalsubject.